Effects of Cell Entrapment in Ca-alginate on the Metabolism of Yeast Saccharomyces cerevisiae

Citation

Galazzo, Jorge Luis (1989) Effects of Cell Entrapment in Ca-alginate on the Metabolism of Yeast Saccharomyces cerevisiae. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/gq59-ka36. https://resolver.caltech.edu/CaltechETD:etd-02022007-144010

Abstract

Saccharomyces cerevisiae cells grown in suspension have been immobilized in calcium-alginate beads. Fermentation rates and intracellular composition have been determined under nongrowing conditions in these Ca-alginate entrapped cells and for identical cells in suspension. Glucose uptake and ethanol and glycerol production are approximately two times faster in immobilized cells than in suspended cells. Intermediate metabolite levels such as fructose-1,6-diphosphate, glucose-6-phosphate and 3-phosphoglycerate have been determined by phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy under glucose fermenting conditions. Results show a different sugar phosphate composition in immobilized cells. Also, at steady-state glucose fermentation, the intracellular pH of entrapped cells is lower as indicated by the chemical shift of the intracellular inorganic phosphorus resonance. Carbon-13 NMR shows an increase in polysaccharide production in immobilized cells.

S. cerevisiae cells grown within a Ca-alginate matrix have a specific growth rate 40% lower that the growth rate of similar cells cultivated in suspension. Alginate-grown cells have been used to compare glucose fermentation under nongrowing conditions in suspended and Ca-entrapped cells. Fermentation rate is higher in immobilized cells than in suspended cells. The observed differences in intracellular components between suspended and immobilized cells are qualitatively similar to the differences observed for cells grown in suspension. Ethanol production rate is 2.7 times faster in immobilized alginate-grown cells than in suspended suspension-grown cells.

These results suggest that cell immobilization is affecting cell metabolism at different levels. Catabolic regulation is altered as indicated in the nongrowing condition experiments. Also, anabolic regulation is altered as suggested by the changes in growth rate observed in cells growing within the immobilization matrix. The combination of these experimental determinations with knowledge of the metabolic pathways involved in S. cerevisiae allows the development of a quantitative in vivo description of most key pathway enzymes involved in yeast glucose catabolism. The evaluation of flux-control coefficients for all these steps indicates that alginate entrapment of suspension-grown cells increases the glucose uptake rate and shifts the step most influencing ethanol production from glucose uptake to phosphofructokinase. In alginate-grown cells, glucose uptake is limiting ethanol production in both suspended and immobilized cells. There is a 5% decrease in the glucose uptake flux-control coefficient of immobilized cells due to an increment in the glucose uptake rate. This increment increases ethanol production by approximately 100%.

An analysis of the anticipated effects of genetic manipulation to improve the ethanol production rate in yeast S. cerevisiae using the framework of the metabolic control theory indicates that in suspended suspension-grown cells the highest improvement is obtained by increasing the activity of glucose transport, whereas in immobilized suspension-grown cells the greatest enhancement is obtained by incrementing the maximum activity of phosphofructokinase. This indicates that the metabolic engineering strategy to be used in order to enhance any properties in the cell will depend not only on the specific microorganism, but also on the environmental conditions in which it is placed. In conditions where several steps share the flux control in a pathway, a substantial enhancement in pathway rate will be possible only if the activities of all those steps are increased simultaneously.

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